Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Erythropoietic protoporphyria (EPP) is an autosomal inherited disease of heme biosynthesis caused by a partial deficiency of the enzyme ferrochelatase. Patients with EPP show only 20-30% normal activity because of mutations in one of the alleles of the ferrochelatase gene. To clarify the molecular mechanisms of this low level of activity, we co-expressed human ferrochelatase carrying His- and HA-tags in a tandem fashion in Escherichia coli. Purification of the His-tag-containing enzyme revealed that the His-enzyme forms an oligomer in association with the HA-enzyme, and analysis by gel-filtration confirmed that the enzyme is a dimer (approximately 80 kDa). Then we expressed homo- and heterodimers composed of the wild-type and engineered mutants of the enzyme (C395Delta, H157A, H263A, H388A) or mutants from EPP patients (I186T, M267I). The levels of homodimeric enzymes produced were low, and the activities of the purified homodimeric mutants were abolished. On the other hand, the heterodimers with wild-type and mutated subunits exhibited potential, but weak, activities without a marked change of Km values for substrates. These results showed that heterodimers containing normal and mutated subunits retain the enzymic activity, which is inconsistent with the hypothesis that ferrochelatase is only active when the dimer contains two normal subunits. Pretreatment at 42 degrees C led to a rapid inactivation of the heterodimeric mutants, indicating instability. Thus, we provide evidence that the instability of the heterodimer containing normal and mutated ferrochelatase as well as the low production levels due to the structural defect of the mutant protein, not the abolishment of the enzymic activity of the heterodimer, causes the weak activity in EPP patients.
Hum Mol Genet 2005 Jan 15
PMID:Ferrochelatase consisting of wild-type and mutated subunits from patients with a dominant-inherited disease, erythropoietic protoporphyria, is an active but unstable dimer. 1557 61

The mitochondrial protein frataxin is emerging as a novel mechanism to promote iron metabolism while also providing anti-oxidant protection. Recombinant frataxin proteins from different species are able to form large molecular assemblies that store Fe(III) as a stable mineral in vitro. Furthermore, monomeric and assembled forms of frataxin donate Fe(II) to the Fe-S cluster scaffold protein IscU, [3Fe-4S]1+ aconitase, and ferrochelatase in vitro. However, little is known about the speciation of frataxin in vivo, and the physiologically relevant form(s) of the protein remains undefined. Here, we report that human heart mitochondria contain frataxin species of increasing negative surface charge and molecular mass, ranging from monomer to polymers of >1 MDa. Moreover, we show that the main partner protein of frataxin, IscU, binds in a stable manner to frataxin oligomers. These results suggest that assembly is a physiologic property of frataxin. Biochemical analyses further reveal that, unlike the prokaryotic and yeast frataxin homologues, which require iron-protein interactions for assembly, human frataxin uses stable subunit-subunit interactions involving a non-conserved amino-terminal region. We propose that human frataxin is a modular protein that depends on self-assembly to accomplish its diverse functions.
J Mol Biol 2005 Jan 21
PMID:Supramolecular assemblies of human frataxin are formed via subunit-subunit interactions mediated by a non-conserved amino-terminal region. 1558 88

In order to compare transcription profiles in cultivars of Malus domestica that are differentially sensitive to apple scab (Venturia inaequalis), two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) method. Subtraction hybridization was performed between cDNAs from uninfected young leaves of the resistant cultivar Remo and the susceptible Elstar. In total, 480 EST clones were obtained: 218 (ELSTAR) clones represent transcripts that are preferentially expressed in Elstar, while the other 262 (REMO) are derived from RNAs that are more highly expressed in Remo. The putative functions of about 50% of the cloned sequences could be identified by sequencing and subsequent homology searches in databases or by dot-blot hybridization to known targets. In the resistant cv. Remo the levels of transcripts encoding a number of proteins related to plant defense (such as beta-1,3-glucanase, ribonuclease-like PR10, cysteine protease inhibitor, endochitinase, ferrochelatase, and ADP-ribosylation factor) or detoxification of reactive oxygen species (such as superoxide dismutase) were highly up-regulated relative to the amounts present in cv. Elstar. Most surprising was the large number of clones derived from mRNAs for metallothioneins of type 3 (91 out of 262) found in the REMO population. The corresponding transcripts were only present in small amounts in young uninfected leaves of the cv. Elstar, but were up-regulated in the susceptible cultivar after inoculation with V. inaequalis. These results indicate that constitutively high-level expression of PR proteins may protect cv. Remo from infection by different plant pathogens.
Mol Genet Genomics 2005 Jun
PMID:Characterization by suppression subtractive hybridization of transcripts that are differentially expressed in leaves of apple scab-resistant and susceptible cultivars of Malus domestica. 1581 49

Several Arabidopsis mutants of the ecotype Dijon were isolated that show resistance to the herbicide acifluorfen, which inactivates protoporphyrinogen oxidase (PPOX), an enzyme of tetrapyrrole biosynthesis. This enzyme provides protoporphyrin for both Mg chelatase and ferrochelatase at the branchpoint, which leads to chlorophyll and heme, respectively. One of the mutations, aci5-3, displays semidominant inheritance. Heterozygous progeny showed yellow-green leaves, while the homozygous seedlings were white and inviable, but could be rescued by supplementing the medium with sugar. Interestingly, the expression of neither of the two forms of PPOX was altered in the mutant, but the rate of synthesis of 5-aminolevulinate, the precursor of all tetrapyrroles, was drastically reduced. Genetic mapping revealed the mutant locus is closely linked to the ch42 marker, which is itself located in the CHLI-1 gene which codes for one of the three subunits of Mg chelatase. The cs mutant also shows a defect in this gene, and test for allelism with aci5-3 confirmed that the two mutations are allelic. Sequencing of the wild type and aci5-3 alleles of CHLI-1 revealed a single base change (G718A), which results in a D240N substitution in the CHLI-1 protein. In the homozygous aci5-3 mutant no CHLI-1 RNA or protein could be detected. Strikingly, CHLH and CHLI-2 transcripts were also absent. This indicates the existence of a feedback-regulatory mechanism that inactivates the genes encoding certain Mg chelatase subunits. The basis for the semidominant inheritance pattern and the relationship between herbicide resistance and modified gene expression is discussed.
Mol Genet Genomics 2005 Jun
PMID:An Arabidopsis mutant that is resistant to the protoporphyrinogen oxidase inhibitor acifluorfen shows regulatory changes in tetrapyrrole biosynthesis. 1581 18

Heme biosynthesis represents one of the most essential metabolic pathways in living organisms, providing the precursors for cytochrome prosthetic groups, photosynthetic pigments, and vitamin B(12). Using genomic data, we have compared the heme pathway in the diatom Thalassiosira pseudonana and the red alga Cyanidioschyzon merolae to those of green algae and higher plants, as well as to those of heterotrophic eukaryotes (fungi, apicomplexans, and animals). Phylogenetic analyses showed the mosaic character of this pathway in photosynthetic eukaryotes. Although most of the algal and plant enzymes showed the expected plastid (cyanobacterial) origin, at least one of them (porphobilinogen deaminase) appears to have a mitochondrial (alpha-proteobacterial) origin. Another enzyme, glutamyl-tRNA synthase, obviously originated in the eukaryotic nucleus. Because all the plastid-targeted sequences consistently form a well-supported cluster, this suggests that genes were either transferred from the primary endosymbiont (cyanobacteria) to the primary host nucleus shortly after the primary endosymbiotic event or replaced with genes from other sources at an equally early time, i.e., before the formation of three primary plastid lineages. The one striking exception to this pattern is ferrochelatase, the enzyme catalyzing the first committed step to heme and bilin pigments. In this case, two red algal sequences do not cluster either with the other plastid sequences or with cyanobacterial sequences and appear to have a proteobacterial origin like that of the apicomplexan parasites Plasmodium and Toxoplasma. Although the heterokonts also acquired their plastid via secondary endosymbiosis from a red alga, the diatom has a typical plastid-cyanobacterial ferrochelatase. We have not found any remnants of the plastidlike heme pathway in the nonphotosynthetic heterokonts Phytophthora ramorum and Phytophthora sojae.
Mol Biol Evol 2005 Dec
PMID:Mosaic origin of the heme biosynthesis pathway in photosynthetic eukaryotes. 1609 70

Insertion of metals into various tetrapyrroles is catalysed by a group of enzymes called chelatases, e.g. nickel, cobalt, magnesium and ferro-chelatase. It has been proposed that catalytic metallation includes distorting the porphyrin substrate by the enzyme towards a transition state-like geometry in which at least one of the pyrrole rings will be available for metal chelation. Here, we present a study of metal insertion into the transition-state inhibitor of protoporphyrin IX ferrochelatase, N-methyl mesoporphyrin (N-MeMP), by time-resolved crystallography and mass spectrometry with and without the presence of ferrochelatase. The results show that metallation of N-MeMP has a very limited effect on the conformation of the residues that participate in porphyrin and metal binding. These findings support theoretical data, which indicate that product release is controlled largely by the strain created by metal insertion into the distorted porphyrin. The results suggest that, similar to non-catalytic metallation of N-MeMP, the ferrochelatase-assisted metallation depends on the ligand exchange rate for the respective metal. Moreover, ferrochelatase catalyses insertion of Cu(II) and Zn(II) into N-MeMP with a rate that is about 20 times faster than non-enzymatic metallation in solution, suggesting that the catalytic strategy of ferrochelatase includes a stage of acceleration of the rate of ligand exchange for the metal substrate. The greater efficiency of N-MeMP metallation by Cu(II), as compared to Zn(II), contrasts with the K(m) values for Zn(II) (17 microM) and Cu(II) (170 microM) obtained for metallation of protoporphyrin IX. We suggest that this difference in metal specificity depends on the type of distortion imposed by the enzyme on protoporphyrin IX, which is different from the intrinsic non-planar distortion of N-MeMP. A mechanism of control of metal specificity by porphyrin distortion may be general for different chelatases, and may have common features with the mechanism of metal specificity in crown ethers.
J Mol Biol 2005 Oct 07
PMID:Metallation of the transition-state inhibitor N-methyl mesoporphyrin by ferrochelatase: implications for the catalytic reaction mechanism. 1614 Mar 24

Deficiency of the frataxin mRNA alters the transcriptome, triggering neuro- and cardiodegeneration in Friedreich's ataxia. We microarrayed murine frataxin-deficient heart tissue, liver tissue and cardiocytes and observed a transcript down-regulation to up-regulation ratio of nearly 2:1 with a mitochondrial localization of transcriptional changes. Combining all mouse and human microarray data for frataxin-deficient cells and tissues, the most consistently decreased transcripts were mitochondrial coproporphyrinogen oxidase (CPOX) of the heme pathway and mature T-cell proliferation 1, a homolog of yeast COX23, which is thought to function as a mitochondrial metallochaperone. Quantitative RT-PCR studies confirmed the significant down-regulation of Isu1, CPOX and ferrochelatase at 10 weeks in mouse hearts. We observed that mutant cells were resistant to aminolevulinate-dependent toxicity, as expected if the heme pathway was inhibited. Consistent with this, we observed increased cellular protoporphyrin IX levels, reduced mitochondrial heme a and heme c levels and reduced activity of cytochrome oxidase, suggesting a defect between protoporphyrin IX and heme a. Fe-chelatase activities were similar in mutants and controls, whereas Zn-chelatase activities were slightly elevated in mutants, supporting the idea of an altered metal-specificity of ferrochelatase. These results suggest that frataxin deficiency causes defects late in the heme pathway. As ataxic symptoms occur in other diseases of heme deficiency, the heme defect we observe in frataxin-deficient cells could be primary to the pathophysiological process.
Hum Mol Genet 2005 Dec 15
PMID:Frataxin deficiency alters heme pathway transcripts and decreases mitochondrial heme metabolites in mammalian cells. 1623 44

Iron and copper homeostasis share common proteins and are therefore closely linked to each other. For example, copper-containing proteins like ceruloplasmin and hephaestin oxidize Fe(2+) during cellular export processes for transport in the circulation bound to transferrin. Indeed, copper deficiency provokes iron metabolism disorders leading to anemia and liver iron accumulation. The aim of the present work was to understand the cross-talk between copper status and iron metabolism. For this purpose we have established dietary copper deficiency in C57BL6 male mice during twelve weeks. Hematological parameters, copper and iron status were evaluated. cDNA microarray studies were performed to investigate gene expression profiles of proteins involved in iron metabolism in the liver, duodenum and spleen. Our results showed that copper deficiency induces microcytic and hypochromic anemia as well as liver iron overload. Gene expression profiles, however, indicate that hepatic and intestinal mRNA expression neither compensates for hepatic iron overload nor the anemia observed in this mouse model. Instead, major modifications of gene expression occurred in the spleen. We observed increased mRNA levels of the transferrin receptors 1 and 2 and of several proteins involved in the heme biosynthesis pathway (ferrochelatase, UroD, UroS,...). These results suggest that copper-deficient mice respond to the deficiency induced anemia by an adaptation leading to an increase in erythrocyte synthesis.
Blood Cells Mol Dis
PMID:Mild copper deficiency alters gene expression of proteins involved in iron metabolism. 1640 11

Genetic analysis has indicated that the system II pathway for c-type cytochrome biogenesis in Bordetella pertussis requires at least four biogenesis proteins (CcsB, CcsA, DsbD and CcsX). In this study, the eight genes (ccmA-H) associated with the system I pathway in Escherichia coli were deleted. Using B. pertussis cytochrome c4 as a reporter for cytochromes c assembly, it is demonstrated that a single fused ccsBA polypeptide can replace the function of the eight system I genes in E. coli. Thus, the CcsB and CcsA membrane complex of system II is likely to possess the haem delivery and periplasmic cytochrome c-haem ligation functions. Using recombinant system II and system I, both under control of IPTG, we have begun to study the capabilities and characteristics of each system in the same organism (E. coli). The ferrochelatase inhibitor N-methylprotoporphyrin was used to modulate haem levels in vivo and it is shown that system I can use endogenous haem at much lower levels than system II. Additionally, while system I encodes a covalently bound haem chaperone (holo-CcmE), no covalent intermediate has been found in system II. It is shown that this allows system I to use holo-CcmE as a haem reservoir, a capability system II does not possess.
Mol Microbiol 2006 May
PMID:Recombinant cytochromes c biogenesis systems I and II and analysis of haem delivery pathways in Escherichia coli. 1662 58

Erythropoietic protoporphyria (EPP) results from an inherited partial deficiency of ferrochelatase, the terminal enzyme of haem biosynthesis. Excess protoporphyrin IX accumulates in erythrocytes, plasma, liver, and skin, which mediates a distinctive form of cutaneous photosensitivity that manifests during childhood. Ferrochelatase is synthesised on cytosolic ribosomes as a preprotein with a cleavable presequence at its amino-terminus. This leader sequence is thought to target ferrochelatase to mitochondria where it is cleaved to produce the active mature protein. In this study, we show that the 62 amino acid leader sequence is sufficient for targeting of a leader sequence-YFP fusion protein to mitochondria. A truncated fusion protein lacking the first 62 amino acids did not target to mitochondria, and formed punctate aggregates in the cytoplasm of cells. This suggests that all the information required for mitochondrial localisation resides within the first 62 amino acid presequence. A missense mutation, P62R, predicted to be located within the ferrochelatase presequence has been identified in a patient with EPP. We hypothesised that this mutation may exert its effect through defective targeting to mitochondria. Our data showed that this mutated full-length ferrochelatase successfully targeted to mitochondria. Interestingly, there was inhibited cleavage of YFP from wild-type and mutant leader sequence fusion proteins. Generation of leader sequence-YFP fusion proteins containing an additional 11 amino acids from the mature protein allowed proteolytic processing to occur. These data suggest that the first 62 amino acids allow targeting to mitochondria but do not contain sufficient information for efficient processing of the protein.
Mol Genet Metab 2006 Nov
PMID:Erythropoietic protoporphyria: a functional analysis of the leader sequence of human ferrochelatase. 1684 98


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